阳离子多肽—硬脂酸嫁接物基因给药系统研究
发布时间:2018-11-05 11:21
【摘要】:阳离子多肽-硬脂酸嫁接物具有较丰富的正电荷,可通过静电作用与质粒DNA (plasmid DNA, pDNA)进行复合,为实现有效的体外基因转染和体内抗肿瘤疗效提供了极大的可能性。本研究构建了阳离子多肽-硬脂酸/pDNA复合物和聚乙二醇修饰多肽-硬脂酸/pDNA复合物基因给药系统,考察了其基本理化性质,体外转染效率,体内抗肿瘤疗效以及细胞内涵体逃逸过程。 阳离子多肽-硬脂酸嫁接物(cationic peptide stearate, STR-Pep)系通过多肽氨基末端的氨基与硬脂酸羧基发生酰胺化反应共轭连接而成,醛基化聚乙二醇则通过醛基与多肽赖氨酸残基上的游离氨基发生反应而生成聚乙二醇修饰的多肽-硬脂酸嫁接物(PEG modified cationic peptide stearate, PEG-STR-Pep)。以核磁共振氢谱确证两种嫁接物的化学结构,芘荧光法测定临界胶束浓度,透射电镜观察胶束的粒径以及形态学特征,噻唑蓝法考察嫁接物细胞毒性。 分别制备STR-Pep/pDNA复合物和PEG-STR-Pep/pDNA复合物,以透射电子显微镜观察复合物的粒径及形态特征,水平凝胶电泳分析嫁接物胶束与DNA的密接程度。以pEGFP-C1作为报告基因,HEK293, SKOV-3, MCF-7, Hela细胞为模型细胞,以荧光倒置显微镜观察嫁接物胶束介导的绿色荧光蛋白表达,并以流式细胞仪定量测定其转染效率。以pGL-3质粒为报告基因,定量考察嫁接物胶束介导的体外转染水平。同时,考察了STR-Pep/pDNA和PEG-STR-Pep/pDNA基因给药系统的体外细胞转染毒性。为了进一步确证多肽中组氨酸的促内涵体逃逸作用,以6-羧基荧光素(FAM荧光)标记pDNA,以Lyso-Tracker标记细胞内内涵体和溶酶体,观察由STR-Pep携载的pDNA在细胞内实现内涵体逃逸的过程。以荷SKOV-3裸鼠为模型动物,以pPEDF (plasmid of pigment epithelium-derivedfactor)为治疗基因,考察STR-Pep/pPEDF基因给药系统的体内抗肿瘤药效学。 因为STR-Pep嫁接物和PEG-STR-Pep嫁接物均具有显著的胶束特性以及较好的基因转染效率,考虑以载药胶束与目的基因结合实现针对肿瘤的协同治疗。本研究以碱基阿霉素(doxorubicin hydrochloride)为模型药物,以透析法制备STR-Pep/DOX和PEG-STR-Pep/DOX载药胶束给药系统;以超滤离心-有机溶剂提取法测定载药胶束中阿霉素的药物含量;以透射电镜观察载药胶束的形态及粒径大小;以SKOV-3、A549、HepG2细胞为模型细胞,评价阿霉素载药胶束的体外药效学。同时,制备了STR-Pep/DOX/pDNA和PEG-STR-Pep/DOX/pDNA复合给药系统,考察其在HepG2细胞中的转染效率。 研究结果表明,通过酰胺化反应和亲和取代反应分别得到了STR-Pep嫁接物和PEG-STR-Pep嫁接物,核磁共振氢谱确证了STR-Pep和PEG-STR-Pep嫁接物的化学组成。STR-Pep和PEG-STR-Pep嫁接物具有良好的理化性质,临界胶束浓度分别为182μg/mL和212μg/mL。透射电镜观察结果显示STR-Pep和PEG-STR-Pep嫁接物胶束粒径大小分别为10-20nm和50-60nm,且具有均匀的球型。STR-Pep和PEG-STR-Pep嫁接物胶束复合pDNA的能力比较强,在水平凝胶电泳实验中,均可以比较低的质量比(w/w=1, w/w=2)完全阻滞pDNA。制备的STR-Pep/pDNA和PEG-STR-Pep/pDNA基因给药系统则成功实现了目的基因的体外转染,STR-Pep/pDNA基因给药系统在正常细胞系HEK293和肿瘤细胞系SKOV-3, MCF-7和HeLa中均达到了较高的转染效率及转染水平,在部分肿瘤细胞系中的转染能力已经非常接近于甚至超越了LipofectamineTM2000。 PEG-STR-Pep/pEGFP基因转染系统则在HEK293细胞系中介导了明显的绿色荧光蛋白表达。在体内抗肿瘤药效学研究中,STR-Pep/pDNA基因给药系统取得了比较好的疗效。同时,STR-Pep和PEG-STR-Pep嫁接物胶束对于阿霉素具有比较高的携载能力,包封率分别达到了54.1%和60.8%,STR-Pep/DOX/pEGFP和PEG-STR-Pep/DOX/pEGFP复合给药系统在体外转染实验中显示了一定的转染能力和发展潜力,相信随着研究的深入,将会取得较好的协同治疗效果。
[Abstract]:The cationic polypeptide-stearic acid graft has a rich positive charge, can be combined with plasmid DNA (pDNA) through electrostatic interaction, thereby providing great possibility for realizing effective in-vitro gene transfection and in vivo anti-tumor treatment effect. In this study, cationic polypeptide-stearic acid/ pDNA complex and polyethylene glycol modified polypeptide-stearic acid/ pDNA complex gene administration system were constructed. Cationic polypeptide-stearic acid grafting (STR-Pep) is linked to the glycolytic reaction by amino acid at amino terminus of polypeptide and stearic acid. and the aldehyde-containing polyethylene glycol generates polyethylene glycol-modified polypeptide-stearic acid grafting (PEG-STR-Pe) through reaction between an aldehyde group and a free amino group on the polypeptide lysine residue. p). The chemical structure of the two kinds of grafts was confirmed by nuclear magnetic resonance hydrogen spectrum, the critical micelle concentration was determined by fluorescence method, the particle size and morphology of the micelles were observed by transmission electron microscope, and the graft cells were examined by means of blue-blue method. Toxicity. STR-Pep/ pDNA complex and PEG-STR-Pep/ pDNA complex were prepared respectively. The particle size and morphology of the complex were observed by transmission electron microscopy. The micelle and DNA of the graft were analyzed by horizontal gel electrophoresis. Using pEGFP-C1 as reporter gene, HEK293, SKOV-3, MCF-7 and Jurkat cells as model cells, the expression of green fluorescent protein mediated by graft micelle was observed by fluorescence inversion microscope, and quantified by flow cytometry. Its transfection efficiency is characterized by that pGL-3 plasmid is used as a reporter gene, and the micelle-mediated body of the graft is quantitatively detected. In addition, STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene were examined in vitro. In order to further confirm the escape of histidine in the polypeptide, pDNA was labeled with 6-streptavidin (FAM fluorescence), and the contents and lysosomes in the cell were labeled with Na-Tracker, and the expression of pDNA carried by STR-Pep in the cells was observed. To study the internal resistance of the STR-Pep/ pPEDF gene to the drug delivery system using the pPEDF-3 nude mouse as the model animal and pPEDF as the model animal. Tumor pharmacodynamics, because STR-Pep grafts and PEG-STR-Pep grafts both have significant micellar properties and better gene transfection efficiency, taking into account the combination of drug-carrying micelles with the target gene to implement the needle In order to prepare STR-Pep/ DOX and PEG-STR-Pep/ DOX micelle administration system by dialysis method, the drug content of doxorubicin in drug-loaded micelle was determined by ultrafiltration centrifugation-organic solvent extraction. The morphology and particle size of drug-loaded micelles were observed by transmission electron microscope. Using SKOV-3, A549 and HepG2 cells as model cells, adriamycin-loaded drug was evaluated. In vitro pharmacodynamics of micelles, STR-Pep/ DOX/ pDNA and PEG-STR-Pep/ DOX/ pDNA composite drug delivery system were prepared. The results showed that STR-Pep grafts and PEG-STR-Pep grafts were obtained by chlorosulfonation reaction and substitution reaction. STR-Pep and PEG-STR were confirmed by nuclear magnetic resonance hydrogen spectroscopy. The chemical composition of Pep grafts, STR-Pep and PEG-STR-Pep grafts have good physical and chemical properties, and the critical micelle concentration is 182. m The particle size of STR-Pep and PEG-STR-Pep was 10-20nm and 50-60, respectively. nm, and has a uniform spherical shape. STR-Pep and PEG-STR-Pep graft micelle complex pDNA has strong ability, and the low mass ratio (w/ w = 1, w/ w) can be compared in the experiment of horizontal gel electrophoresis. (2) Complete blocking of pDNA. STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene delivery systems were successfully transfected in vitro, and the STR-Pep/ pDNA gene administration system was found in normal cell line HEK293 and tumor cell line SKOV-3, MCF-7 and HeLa. High transfection efficiency and transfection level, the ability to transfect in some tumor cell lines has been very close to even more than Josephine. MineTM2000. PEG-STR-Pep/ pEGFP gene transfection system is mediated in HEK293 cell line. Significant green fluorescent protein expression. In vivo antitumor pharmacodynamic studies, STR-Pep/ pDNA gene was administered. At the same time, STR-Pep and PEG-STR-Pep graft micelles had higher carrying capacity for doxorubicin, and the entrapment efficiency reached 54. 1% and 60. 8% respectively. STR-Pep/ DOX/ pEGFP and PEG-STR-Pep/ DOX/ pEGFP composite drug delivery systems showed certain transfection ability and development potential in vitro transfection experiments. Force, believe that as the study goes deep, it will
【学位授予单位】:浙江大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R94
本文编号:2311903
[Abstract]:The cationic polypeptide-stearic acid graft has a rich positive charge, can be combined with plasmid DNA (pDNA) through electrostatic interaction, thereby providing great possibility for realizing effective in-vitro gene transfection and in vivo anti-tumor treatment effect. In this study, cationic polypeptide-stearic acid/ pDNA complex and polyethylene glycol modified polypeptide-stearic acid/ pDNA complex gene administration system were constructed. Cationic polypeptide-stearic acid grafting (STR-Pep) is linked to the glycolytic reaction by amino acid at amino terminus of polypeptide and stearic acid. and the aldehyde-containing polyethylene glycol generates polyethylene glycol-modified polypeptide-stearic acid grafting (PEG-STR-Pe) through reaction between an aldehyde group and a free amino group on the polypeptide lysine residue. p). The chemical structure of the two kinds of grafts was confirmed by nuclear magnetic resonance hydrogen spectrum, the critical micelle concentration was determined by fluorescence method, the particle size and morphology of the micelles were observed by transmission electron microscope, and the graft cells were examined by means of blue-blue method. Toxicity. STR-Pep/ pDNA complex and PEG-STR-Pep/ pDNA complex were prepared respectively. The particle size and morphology of the complex were observed by transmission electron microscopy. The micelle and DNA of the graft were analyzed by horizontal gel electrophoresis. Using pEGFP-C1 as reporter gene, HEK293, SKOV-3, MCF-7 and Jurkat cells as model cells, the expression of green fluorescent protein mediated by graft micelle was observed by fluorescence inversion microscope, and quantified by flow cytometry. Its transfection efficiency is characterized by that pGL-3 plasmid is used as a reporter gene, and the micelle-mediated body of the graft is quantitatively detected. In addition, STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene were examined in vitro. In order to further confirm the escape of histidine in the polypeptide, pDNA was labeled with 6-streptavidin (FAM fluorescence), and the contents and lysosomes in the cell were labeled with Na-Tracker, and the expression of pDNA carried by STR-Pep in the cells was observed. To study the internal resistance of the STR-Pep/ pPEDF gene to the drug delivery system using the pPEDF-3 nude mouse as the model animal and pPEDF as the model animal. Tumor pharmacodynamics, because STR-Pep grafts and PEG-STR-Pep grafts both have significant micellar properties and better gene transfection efficiency, taking into account the combination of drug-carrying micelles with the target gene to implement the needle In order to prepare STR-Pep/ DOX and PEG-STR-Pep/ DOX micelle administration system by dialysis method, the drug content of doxorubicin in drug-loaded micelle was determined by ultrafiltration centrifugation-organic solvent extraction. The morphology and particle size of drug-loaded micelles were observed by transmission electron microscope. Using SKOV-3, A549 and HepG2 cells as model cells, adriamycin-loaded drug was evaluated. In vitro pharmacodynamics of micelles, STR-Pep/ DOX/ pDNA and PEG-STR-Pep/ DOX/ pDNA composite drug delivery system were prepared. The results showed that STR-Pep grafts and PEG-STR-Pep grafts were obtained by chlorosulfonation reaction and substitution reaction. STR-Pep and PEG-STR were confirmed by nuclear magnetic resonance hydrogen spectroscopy. The chemical composition of Pep grafts, STR-Pep and PEG-STR-Pep grafts have good physical and chemical properties, and the critical micelle concentration is 182. m The particle size of STR-Pep and PEG-STR-Pep was 10-20nm and 50-60, respectively. nm, and has a uniform spherical shape. STR-Pep and PEG-STR-Pep graft micelle complex pDNA has strong ability, and the low mass ratio (w/ w = 1, w/ w) can be compared in the experiment of horizontal gel electrophoresis. (2) Complete blocking of pDNA. STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene delivery systems were successfully transfected in vitro, and the STR-Pep/ pDNA gene administration system was found in normal cell line HEK293 and tumor cell line SKOV-3, MCF-7 and HeLa. High transfection efficiency and transfection level, the ability to transfect in some tumor cell lines has been very close to even more than Josephine. MineTM2000. PEG-STR-Pep/ pEGFP gene transfection system is mediated in HEK293 cell line. Significant green fluorescent protein expression. In vivo antitumor pharmacodynamic studies, STR-Pep/ pDNA gene was administered. At the same time, STR-Pep and PEG-STR-Pep graft micelles had higher carrying capacity for doxorubicin, and the entrapment efficiency reached 54. 1% and 60. 8% respectively. STR-Pep/ DOX/ pEGFP and PEG-STR-Pep/ DOX/ pEGFP composite drug delivery systems showed certain transfection ability and development potential in vitro transfection experiments. Force, believe that as the study goes deep, it will
【学位授予单位】:浙江大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R94
【参考文献】
相关期刊论文 前1条
1 李超;韩金路;王玉刚;孙英凯;;流式细胞仪的工作原理及应用[J];中国实用医药;2009年20期
,本文编号:2311903
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